Cryopreservation of Hematopoietic Stem Cells

Cryopreservation of Hematopoietic Stem Cells

David Berz*, Elise M. McCormack, Eric S. Winer, Gerald A. Colvin, and Peter J. QuesenberryRoger Williams Medical Center, Bone Marrow Transplant Unit, East Wing, Providence, RhodeIsland

AbstractStem cell transplantation represents a critical approach for the treatment of many malignant and non-malignant diseases. The foundation for these approaches is the ability to cryopreserve marrow cellsfor future use. This technique is routinely employed in all autologous settings and is critical for cordblood transplantation. A variety of cryopreservatives have been used with multiple freezing andthawing techniques as outlined in the later chapters. Freezing efficiency has been proven repeatedlyand the ability of long-term stored marrow to repopulate has been established. Standard approachesoutlined here are used in many labs as the field continues to evolve.

Keywordsstem cell; marrow; cryopreservative; freezing methods

INTRODUCTIONThe role of hematopoietic stem cell transplantation in the treatment of hematologic andnonhematologic malignancies is rapidly expanding. In certain situations fresh stem cells canbe employed in the setting of allogeneic transplantation. If the transfer from donor to recipientcan be established within 72 hr, protocols for preliminary storage at suprafreezing temperaturesare in place. However, the current therapeutic strategies demand that the progenitor cells arecryopreserved for virtually all autologous and many allogeneic transplants. This strategy hasbeen proven to be safe and not associated with significant adverse outcomes regarding failureto engraft, graft versus host disease (GVHD), or engraftment failure [1].

The cryopreservation process is of importance for all types of stem cell collection, but isperhaps particularly critical for umbilical cord blood (UCB). The actual transplant is hereharvested at the time of birth and used at a later point in time for a yet, at the time point of theharvest, often indeterminate recipient. The UCB is usually stored by either public or privatecord blood banks. Public cord blood banks are usually nonprofit organizations, which offer thedonor unit to matching recipients via national or international registries to potential recipientsin need [2]. Cord blood banks store a donor specimen for the donor or in the case of publicbanks for an unknown recipient for an indeterminate time span. There are now about 170,000frozen units in 37 cord-blood registries in 21 countries. Two thousand nine hundred units havebeen transplanted to date, with adults having received about one third of those units [3].

The cryopreservation process entails the following general components:

1. Harvesting of the donor cells, which entails the actual collection of the specimen andthe reduction of bulk.

*Correspondence to: David Berz, M.D., Ph.D., Roger Williams Medical Center, Bone Marrow Transplant Unit, East Wing, 825Chalkstone Avenue, Providence, RI 02908-4735. E-mail: [email protected] sponsor: NIH; Contract grant numbers: 1P20RR018757, 5R01, DK61858, 5KO DK064980.

NIH Public AccessAuthor ManuscriptAm J Hematol. Author manuscript; available in PMC 2007 November 12.

Published in final edited form as:Am J Hematol. 2007 June ; 82(6): 463–472.

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2. Addition of cryopreservatives

3. The actual freezing procedure

4. Assessment of the viability of the frozen unit after about 72 hr

5. The thawing procedure

6. The washing and conditioning of the donor unit prior to transplant

No single cryopreservation method has been universally used. Variations in technique occurbetween different transplant centers. Our review revealed that slight changes have beenobserved over the last 15 years [4]. At our institution we use a standardized NIH protocol forthe preservation of allogeneic and autologous peripheral hematopoietic stem cell and bonemarrow transplant specimens.

We collect Hematopoietic Progenitor Stem Cells in a minimally manipulated fashion as definedby the Foundation for the Accreditation of Hematopoietic Cell Therapy (FAHCT) with aminimal cell dose of at least 2.5 × 106–5.0 × 106 CD34(+) cells/kg body weight, as currentlyconsidered standard [5]. The specimen is then centrifuged to develop the cell rich pellet. Inautologous transplants donor plasma is used. The supernatant out of this cryoprecipitationprocess is used for the reliquidification of the pellet cells, after the addition of a solution ofheparinized Plasmalyte solution and 10% DMSO (Dimethylsulfoxide). This usually eventuatesinto a cellular concentration of 500 × 10−6 cells in the cryopreservate. We store the bone marrowor peripheral blood stem cell product at initially −4°C [6]. Then the sample is frozen down tothe target temperature of −156°C (when stored in the vapor phase) to −196°C (when stored inthe liquid phase), depending on where in the container the specimen is stored. To guaranteethe integrity of the donor unit prior to myeloablative therapy, a viability reassessment of theunit is performed using a Trypan Blue assay, and if this is equivocal a propidium iodide assay.Prior to the actual stem cell infusion the sample is rapidly thawed in a 37°C water bath.

Several elements of the cryopreservative procedure are still matter of debate. Algorithmsdiffering in freezing temperature, freezing rate, cryopreservatives, durability, thawingtemperature, and thawing rate have been published. This article will review the status of theliterature regarding some of those elements [4,7,8].

TEMPERATUREThe temperatures used for the cryopresevation of hemopoeitic stem cells over the past fifteenyears has been −196, −156, or −80°C, reflecting the storage temperatures in liquid and vaporphase nitrogen and in cryopreservation mechanical freezers, respectively. The development ofthe use of cryopreservative temperatures was from lower temperatures of around −196°C inthe 1980s to around −80°C in the 1990s [2,9–16]. For umbilical cord isolated stem cells similartrends are observed. The standard temperatures currently in use are −196 to −80°C [17–19].Secondary to the recent reports about the spread of infectious agents (i.e., aspergillus as wellas viral spread), through the liquid phase of the nitrogen tanks, the currently recommendedoptimal storage conditions are in the vapor nitrogen phase, at −156°C. Mechanical freezersmight represent a viable alternative.

Also, several studies examined the possibility to store HSCs at suprafreezing temperature, at4°C. A preclinical study that examined PBSCs mobilized in autologous plasma with post-storage clonogenic and viability assays suggested that a storage up to five days is safe [20]. Asmall case series by Ruiz-Arguelles et al. successfully used PBPCs after 96 hr storage at 4°Cfor rescue after high dose chemotherapy [21].

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FREEZING RATEThe rate of freezing was widely debated in the literature. The controlled rate freezing techniqueis still considered standard, mainly due to the fact that the heat liberation at the transition oreutactic point (about 4°C) is deemed detrimental to the stem cell population. At this point thewater molecules within the frozen unit are in a precise molecular order, what eventuates in thethermodynamic liberation of fusion heat.

In controlled rate freezing, the concentrated stem cells are frozen down at a rate of 1–2°C/minup to a temperature point of about −40°C. Then, the freezing process down to a target of −120°C is performed at a faster pace, about 3–5°C/min. For umbilical cord stem cells, bone marrow,and PBSCs the controlled rate freezing process is considered standard [22–24], and was indifferent reports found to be superior to uncontrolled freezing approaches. This procedure istime consuming and requires staff with a specific expertise. Hence, the use of uncontrolled ratefreezing in which the specimen is first cooled down to −4°C and then directly deposited intoa freezer at −80°C or put into liquid phase nitrogen has been evaluated. Several reports [9,12,13] established that the uncontrolled method is safe and reveals comparable results to thecontrolled rate process for BM and PBSCs. A controlled study performed by Perez-Oteyza etal. [14] showed that the controlled and uncontrolled rate freezing approach are comparable interms of viability testing and that only a statistically significant decrease in the CFU-GMclonality assay could be detected in the uncontrolled freezing situation. Recent studiessuggested that uncontrolled freezing is also a viable approach for UCB stem cells [25,26]. Noconsensus exists about the relevance of the compensation for fusion heat during the freezingprocedure [6]. However, a study of Balint et al. outlined the importance of this intervention,comparing five different freezing protocols [6]. The protocols using a five step controlledfreezing approach compensating for the fusion heat achieved better post thawing viability.

DURABILITYThe actual durability, defined as the time that stem cells can be preserved, is still unclear. Theviability of the stem cells in cryopreservation has been questioned in different studies after atime course of cryopreservation of 6 months [10]. Further studies have demonstrated aprolonged freezing time with complete preservation of stem cell function.

Several substitute assays are used to estimate the functional hematopoietic reconstitutivecapacity of the first frozen and then thawed specimens. While BFU-E and CFU-GM appear tobe compromised earlier in the course of cryopreservation, the recovery of nucleated cells (NC)and CD34+ cells and the actual engraftment in NOD/SCID mice seems to be preserved for alonger period of time [2,9,12,14 15,27,28]. Those observations have been initially made in bonemarrow and PBSC, and similar observations have been made with UBC stem cells [18,29–33]. The NOD/SCID mouse assay is currently considered the most valuable assay to assess thefunctionality for hematologic recovery of HSC preparations [34–37], but is not routinelypractical. After Kobylka et al. [38] and Mugishima et al. [39] proved the durability after 12and 15 years with flow cytometric and clonogenic assays, respectively, and Broxmeyer et al.[30] performed a reassessment of his long-term preserved CB units, a durability of up to 15years was established by using hematopoietic reconstitution in NOD/SCID mice. Clinicalvalidity of preclinical studies was documented in anecdotal reports when successful trilineageengraftment was achieved with BM, stored for 7 years [26]. A systematic review evaluatingthe combined experience of the Brigham and Women’s Hospital and the EBMT Group [11]noticed that HSC can be effectively cryopreserved for up to 11 years. A retrospective studyfrom Seattle revealed full trilineage recovery in patients receiving HSC, stored for up to 7.8years without consistent detrimental effects [27].

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CELL CONCENTRATIONReinfusion of cryopreserved cells has been associated with varying toxicities, which werepartially attributed to the total volume and the cryopreservatives in the solution [7,8,40].Concern was raised in the past that a high cell concentration in the cryopreservate can eventuatein toxicity to the cells. Hence, the initially proposed concentration of cryopreserved cells wassuggested to be not over 2 × 10−7/ml NC [11,41]. This would eventuate in a cryopreservationvolume of about 7 l [10] per patient. The storage space needed and the labor to wash the graftprior to reinfusion would be immense [4].

After initial murine models were found safe, Rowley et al. established that high cellconcentrations (up to 5.6 × 10−8 cells/ml) in the cryopreservate are well tolerated, not associatedwith significant adverse effect to the cells, and resulted in good clinical outcomes [4]. Similarconclusions were drawn from subsequent studies by Kawano et al. [42] and Cabezudo et al.[43]. For practical purposes a cell concentration of 200 × 10−6/ml appears achievable [4,44,45].

CRYOPRESERVATIVESCryopreservatives are necessary additives to stem cell concentrates, since they inhibit theformation of intra and extracellular crystals and hence cell death. The standard cryoprotectantis DMSO, which prevents freezing damage to living cells [46]. It was initially introduced intomedical use as an anti-inflammatory reagent and is still occasionally used in auto-immunedisorders [47,48]. Usually it is used at concentrations of 10% combined with normal salineand serum albumin [2,9,13,49]. This was established to be a safe and non-stem cell toxic agent[49]. However, DMSO is associated with a clinically significant side effect profile. Nausea,vomiting, and abdominal cramps occur in about half of all the cases [50]. Other side effectsencompass cardiovascular [7], respiratory [51,52], CNS [8,53–55], renal, hemolytic [56], andhepatotoxic presentations. Case fatalities attributed to DMSO toxicities have been reported[57].

A recent multinational questionnaire based survey, including data from 97 EBMT transplantcenters, revealed that DMSO related toxicities other than nausea and vomiting are observed inabout one out of 50 transplants with a mean incidence of 2.2% of all administered units.Cardiovascular side effects were the most frequently observed group of adverse eventswitnessed in 27% of the participating centers. Respiratory events were observed in 17%, CNStoxicities, including seizures, in 5%, and renal adverse effects in 5% [58].

Based on these toxicity considerations, newer approaches have been tried. Lower dosages ofDMSO, varying from 2.2 to 6% [9,10,12,59,60] have been established to be efficacious in bonemarrow and PBSCs as well as for UCB. On the contrary, a Yugoslavian study compared the10% DMSO concentration to lower concentrations with different freezing rates. The 10%DMSO cryopreservation proved to be superior to lower concentrations in this in vitro study[6]. To enhance the effect of the cryopreservative, the combination of DMSO and theextracellular cryoprotectant hydroxyethyl starch (HES) has been used with success in PBSCsand bone marrow grafts [12,13] and UCB cells.

Alternative preservation methods for cryopreservation are propylene glycol, a combination ofalpha tocopherol, catalase, and ascorbic acid and the glucose dimer trehalose as intra andextracellular cryoprotectant [28,32,61].

Interesting preclinical data from Germany suggests that activation of caspases, particularlyduring the thawing process, can induce apoptosis and hence contribute to the cryoinjury to

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transplant grafts. Addition of the caspase inhibitor zVAD-fmk as cryopreservative presents anintriguing future perspective [62].

THAWINGSeveral techniques for the thawing procedure have been proposed. The standard method iswarming in a water bath at 37°C until all ice crystals disappear [13]. A German study comparedthe thawing of cryopreserved units in a warm water bath with dry heat applied by gel pads at37°C. The viability and clonogenic potential were comparable, with a trend towards lessinfectious contamination in the dry method [63]. Different studies examined the preservationof function when thawed units were incubated at 0–37°C [13,31]. No significant differenceswere detected in a study by Yang et al. who compared an incubation of the thawed unit at 0,20, and 37°C for 20 min [31]. The used cryopreservative proved to be nontoxic to the stemcells during the cryopreservation process, as already established by previous studies [4,49].Reducing the DMSO content at thawing temperature is an intriguing concept, because of theclinical toxicity profile of this cryopreservative. Hence, the effect of reducing the DMSOcontent in the thawing solution by virtue of washing or dilution has been explored [59,64].Minor or no effect on the stem cell viability has been observed. An automated method to washout the cryopreservative has proven feasible in pre-clinical models [65].

WASHING PROCEDUREFor stem cells of cord, peripheral blood, and bone marrow origin, the process of washing outthe cryopreservative after the thawing can still be considered standard [19,32], since the DMSOis assumed to have toxic effect on the stem cells. This has been questioned by several morerecent reports, which suggested stem cell resistance against DMSO exposure [13,49]. The washout of the most popular cryopreservative has conceivable benefits for the recipient, i.e.reduction of toxicity, since the degree of DMSO toxicity is proportional to the amount ofDMSO contained in the infused stem cell solution [66]. It was also suggested that wash out ofDMSO can enhance engraftment [67]. This has been disputed [68].

The current standard washing protocol follows the New York Blood Center protocol [19], inwhich the two step dilution of the thawed stem cell unit with 2.5% human serum albumin and5% dextran 40 is followed by centrifugation at 10°C for 10 min. The supernatant is thenremoved and HSA and dextran solution is again added twice to a final DMSO concentrationof less then 1.7%. The washed solution is infused as soon as possible. Although this procedurehas been established to be safe and associated with a reasonable recovery of NC and progenitorassays [69], it is also very labor intensive and not free of cell loss [70,71]. Recently newautomated cell washing devices have been introduced with promising results [50,65,72,73].

CONTAINERS FOR STORAGEThe International Society for Cellular Therapy (ISCT) described on its supplier informationwebsite for cryocontainers nine different cryostorage container products. Six of them areEthinyl Vinyl Acetate (EVA) based, usually gamma irradiated.

Trademarks are• Cryocyte/Baxter• CellFlex/Maco Pharma• Cell Freeze™ Charter Medical• Pall Medical Freezing Bag 791-05

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• Cryostore EVA/Origen Biomedical Inc.• Thermogenesis Corp./Freezing bag 80346-0

Other, not EVA based products are• American Fluoroseal/FEP(Teflon)• Fresenius Hemocare/Hemofreeze(Teflon,Kaplon)• Origen Biomedical Inc./Permalife Bag, FEP/Polyimide

Other approaches have been undertaken. A Czech group published their successful experiencewith a stainless steel cryopreservation container specifically designed for PBSC [74]. In theUS, the most commonly used cryocontainer is an EVA freezing bag (Yang, 2005 [75]), [76].

The use of specific containers, PVC and polyolefin plastic bags and polyethelene cryostoragevials, achieved different results regarding the viability of the stored specimen. PVC andpolyolefin bags achieved satisfactory results, while polyethelene cryostorage vials did not inone study [2]. A group from Boston suggested that polyolefin cryobags achieve a longerfunctional duration than PVC bags.

INFECTIOUS CONSIDERATIONSMicrobial contamination of transplants represents a significant hazard to the severelyimmunosuppressed recipient. The FDA estimates that seven transplant related deaths per yearcould be avoided by elimination of infection related to donor cell infusions [77]. The overalldemonstrated microbiological contamination rates are 0–4.5% [27,78–83].

The major parts of the cultured bacteria are skin flora and commensal bacteria. The remainderis mainly made up out of enteric bacteria. Of note is that bone marrow derived stem cellcryounits are much more likely to be contaminated by pathogens, which is explained by theharvesting process. The rates of contamination between PBSC and bone marrow differsignificantly by up to a factor of sixteen (0.23% for PBSC and 3.8% for BM) [84].

Table I displays the incidence with which different pathogens were cultured from donor unitsin four different studies addressing the bacterial contamination of stem cell products [78,82–84]:

The cryopreservation process was associated with reduction of detectable microorganisms. Inone German study the detection of Staphylococcus epidermidis was reduced by an average of9.3% and Escherichia coli by 18.1% [85]. Also, several studies reported the occurrence ofpositive cultures post thawing [27,83,86]. This suggests the risk of contamination of the culturebottles (i.e., not induced by the donor graft).

The incidence of severe sepsis upon infusion of stem cells, which cultured positive forcommensals and skin flora bacteria, is low and most of the febrile episodes developing aftertheir infusion are treatable with antibiotics [83].

The stability of viruses in liquid nitrogen has been documented [87]. An English sourcepublished an epidemic outbreak of hepatitis B in autologous bone marrow recipients [88],which was subsequently linked by nucleotide sequence analysis to another cryopreservativestored in the same container [89]. Subsequent analysis of debris in the liquid nitrogen phaseof the same container demonstrated spread of the pathogen via the liquid phase. Similaroutbreaks have been reported [90]. To prevent such incidences we store infectious conservesseparately and provide protective sleeves around the cryopreservative bags, as reported to beefficacious previously [91].

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To prevent infectious complications by the infusion of donor stem cells the following measuresshould be employed:

1. Processing in clean areas and scrupulous microbiologic monitoring of all stages ofthe stem cell preservation procedure according to current standards [92,93].

2. Detection of microbiologic contamination prior to infusion. This has beensuccessfully done in an automated manner for other blood products [86].

3. Screening of donors, even in the autologous setting as circulated in regular guidelines[92]. Upon detection of an infectious graft there should be separate or protectedstorage [91].

EMBRYONIC STEM CELLSEmbryonic stem cells portray a different biologic behavior under cryopreservation. Becauseof the enormous potential of Embryonic Stem (ES) cells for transplantation therapy, recentstudies have evaluated the manner in which these fragile cells are stored. One difficulty withthe cryopreservation of these cells is their extreme fragility resulting in poor survival of thecells under standard freezing procedures, usually in the range of 1% [93]. Not only do the cellshave a poor yield with the standard freezing protocols, the cells are also induced intodifferentiation. Ware et al. investigated a method using a very slow controlled rate freezingprocedure and 10% DMSO. Along with the very slow freezing rate, a rapid thaw was found tobe critical for a successful storage [93]. A study from Wisconsin identified HES (hydroxy ethylstarch) as a valuable cryoadditive during slow rate freezing and vitrification procedures [94].Some methods have been derived in which the ES cells are frozen in 24-well plates withminimal media and 10% DMSO at −80°C. The importance of rapid thawing by adding minimalmedia at body temperature was emphasized. In a study by Ure et al., all 227 clones tested grewsuccessfully, although molecular and phenotypic studies were not done in this instance to provethe cell lineage [95]. Adams et al. successfully cryopreserved primary hepatocytes in aUniversity of Wisconsin solution containing FBS and DMSO for 8 months with thepreservation of viability and key phenotypic properties [96]. A recent preclinical study byMilosevic et al. highlighted the role for caspase inhibitors as additives to cryopreservative inembryonic murine neural precursors, an approach that was previously undertaken inhematopoietic precursors [62]. A 60–70% viability was achieved by adding the caspaseinhibitor zVAD-fmk to different cryopreservatives after five days [99].

One theory as to the cause of cell death is ice crystal formation in the cytoplasm during freezing[98]. The process of vitrification makes attempts of freezing the ES cells while avoiding theice formation. Reubinoff was the first to implement this method using an open pulled strawvitrification method, which had previously been successful in the cryopreservation of embryos.This procedure, which evaluated the more fragile human ES cells, proved a 100% viability ofthe ES cell clumps that all generated colonies compared to a 70% recovery post thaw and 16%differentiation using standard methods [99]. The test cells also had normal karyotypes, OCT-4expression, and developed teratomas in xenografts of SCID mice [100]. Since this experiment,others have looked at closed seal straw vitrification or alternative freezing media and asimplified vitrification method [100,101] that showed similar results.

FUNCTIONAL SUBSTITUTE ASSAYSThe most commonly used clonogenic assays are CFU-Sd12 [6], a murine assay, MRA (CFU-GM) [6,27,32,38,66,102], CFU-GEMM [6,103], BFU-E [27,38,66,103], and the long termculture initiating cell [104] (LTC-IC) assay (Fig. 1). Still, in spite of the availability of theseassays to quantitatively and qualitatively assess the clonogenic potential of the hemopoietic

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cell in suspension, the eventual evaluation of the engraftment potential relies on the evidenceof hematopoietic reconstitution in myeloablated mammals.

Different techniques that have been used for human cells are cell counting for NC [32,66,105] and CD34+ cells [61,105], tryptan blue exclusion for viability [94,103], 7-Aminoactinomycin [29,31], engraftment in NOD/SCID mice, and clonogenic assays [27].

Though no absolute consensus is reached as to the optimal method to assess the functionalityof a donor graft, several substitute assays have been used to estimate the functionalhematopoietic reconstitutive capacity of the first frozen and then thawed specimen. The broadcategories of assays used for this purpose are cell counting assays, viability assays, clonogenicexperiments, and the engraftment of donor cells in NOD/SCID mice.

1. Cell counting assays Count of NC [32,66,105]Flow cytometry of CD34+ cells [61,105]

2. Viability assays Tryptan blue [93,103]7-Aminoactinomycin [29,31]Propidium iodide [106,107]

3. Clonogenic assays CFU-Sd12 [6] assay in miceCFU-GM [6,27,32,38,66,102]CFU-GEMM [6,32]BFU-E [27,38,66,102]LTC-IC [104]

4. Direct engraftment experiments NOD/SCID mice [2,9,12,14,15,27]

While the BFU-E and CFU-GM appear to be compromised earlier in the course ofcryopreservation, the recovery of NC and CD 34+ cells and the actual engraftment in NOD/SCID mice seems to be preserved for a longer period of time [2,9,12,14,15,27,28]. Theseobservations have been made in bone marrow and PBSC, and for UBC the same observationswere made [18,29–33].

Yang et al. evaluated two different functional assays, the CD 34 count and the CFU-GM, bycorrelating the pre and post thawing assay outcome with engraftment in 52 patients. The preand post thawing assay correlated well with each other and with the actual clinical engraftment(Yang et al., 2005, [24]). A general summary of a standard cryopreservation technique ispresented in Figure 2.

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Fig 1.Clonogenic assays. Cells are plated in soft agar and incubated. At set time periods, the cellcolonies are counted; this provides an in vitro surrogate of hematopoietic reconstitutionpotential. (A) CFU/GM (B) CFU/GEMMA.

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Fig 2.The cryopreservation process for bone marrow, peripheral blood stem cells, and UCB.

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TABLE IOrganisms Cultured/Overall Incidence of Positive Cultures

Organisms cultured Overall incidence of positive cultures (%)

Staph. epidermidis and other coagulase negative Staphylococcus (CNS) 3–11.7Propionibacterium acni 0.6–2.2Staphylococcus aureus 0–1.6Bacillus cereus and other Bacillus spec. 0.06–0.35Pseudomonas spec.(aeruginosa, putida and fluoresces) 0.1–0.8Corynebacterium spec. 0–0.3Aspergillus fumigatus 0–0.3Mixed cultures 0.1–1.6


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Cryopreservation of Hematopoietic Stem Cells